All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
A major problem associated for the commercialization of transgenic cultivars in highly heterozygous crops is the segregation of transgenes during seed production (Conner and Christey Biocontrol Science and Technology 4:463-473 (1994)). In order to develop a cultivar involving crosses between heterozygous individuals (e.g.: asparagus, forage brassicas, pasture species, forest trees, etc.), it will be necessary to intermate individuals heterozygous for transgenes. In many open pollinated or synthetic cultivars this will usually involve the intermating of several transgenic lines independently derived for different individual plants. This will involve the parents of synthetic cultivars, or a sufficient number of different individuals to maintain an effective population size to avoid inbreeding depression/genetic drift within the population. The transgenic individuals utilized in producing transgenic synthetic populations may arise from single event transformations of a single plant. When this is the case, the transgene could be introduced into a synthetic population, such as an alfalfa synthetic population, by making multiple crosses of the individual transgenic alfalfa plant with a number of different non-transgenic alfalfa plants from one or more alfalfa lines. Alternatively, since the transgenic individuals to be intermated may be derived from independently derived transformed plants, the transgenes may be located at different loci. The resulting intermated progeny will therefore be segregating at all the loci and the transgenic traits will have a "quantitative basis" (Conner and Christey, supra). As discussed immediately below, the prior art has failed to address the segregation and consequent loss of transgenes in open pollinated and synthetic populations.
U.S. Pat. No. 5,254,801 discloses methods whereby plant cells and whole plants can be genetically modified so as to selectively induce cellular lethality using heterologous dominant, conditionally lethal genes in combination with selected protoxin compounds. The methods are for inducing male sterility for the hybrid seed production, including alfalfa, canola, and oil seed rape. This patent fails to disclose a method of producing heterologous plants which utilizes a transgene coding for resistance to a specific phytotoxin.
U.S. Pat. No. 5,278,057 describes a method of producing plants with a marker closely linked to a target locus, in particular a nuclear male sterile target locus. The method involves transformation of a group of plants in order to introduce a marker into each plant, and isolation of a plant with the marker closely linked to a target locus. The markers include visible markers and dominant conditional lethal markers (e.g., antibiotic resistance or herbicide resistance). The method is of particular use for hybrid seed production of any crop plant where the target locus is a nuclear male sterile locus, including rapeseed, alfalfa, clover, cole crops or Brassica oleracea.
U.S. Pat. No. 5,426,041 discloses a method for seed preparation which comprises:
a) crossing a male sterile plant and a second plant which is male fertile, PA1 b) obtaining seed of said male sterile plant, wherein the seed has integrated into its genome: PA1 1. Herbicide resistance: Roundup.RTM., Arsenal.RTM., and sulfonyl urea herbicides such as chlorsulfuron. PA1 2. Insect resistance: Use of Bt or other genes conferring resistance to important insect pests such as Lygus (Lygus sp.), alfalfa caterpillar (Colias sp.), and alfalfa weevil (Hypera sp.). PA1 3. Forage quality: Increasing rumen escape (bypass) protein, which would improve feed efficiency and utilization; decreased lignin content, which would improve digestibility and animal performance. PA1 4. Physiological: Elimination of leaf senescence, would improve yield and quality as well as decrease leaf disease development (see, U.S. Pat. No. 5,689,042).
1) a first recombinant DNA molecule having a first DNA sequence which encodes a first gene product and a first promoter which is capable of regulating the expression of said first DNA sequence; and, PA2 2) a second recombinant DNA molecule which contains a second DNA sequence which encodes a second gene product and a second promoter which is capable of regulating the expression of said second DNA sequence.
The first and second gene products cooperate to selectively interfere with the function and/or development of cells of a plant that are critical to pollen formation and/or function of a plant grown from said seed, such that any plant grown from the seed is substantially male sterile.
More specifically, the '041 patent further teaches a procedure to produce hybrid seed which includes using an IamS/IamH genetic system. The procedure can include linking the IamH gene to a gene for herbicide resistance so that the herbicide can be used for the roguing of the plant line A1; and, that herbicide application takes place after flowering and will kill the A1 so that only seed that has the genotype A1/A2 is produced. The A1/A2 seed is substantially 100% male sterile and can be pollinated with a male fertile line leading to commercial hybrid seed.
U.S. Pat. No. 5,633,441 is directed to plants comprising female-sterility DNA encoding a protein or polypeptide such as barnase which, when produced in the cells of the plant, kills or significantly disturbs the metabolism, functioning or development of the cells. The foreign DNA also comprises a first promoter which directs expression of the female-sterility DNA selectively in style cells, stigma cells or style and stigma cells of the female reproductive organs of the plants. The first promoter does not direct detectable expression of the female sterility DNA in the ovule or in other parts of the plant so that the plant remains male-fertile. The female-sterility DNA is in the same transcriptional unit as and under the control of the first promoter. More specifically, the '441 patent discloses a foreign chimaeric DNA sequence that comprises the female-sterility DNA and a first promoter and that can also comprise a marker DNA and a second promoter. Preferred markers include herbicide tolerance or resistance genes.
The '441 patent further discloses a process for producing hybrid seeds, which grow into hybrid plants, by crossing: 1) the female-sterile plant of this invention which may include, in its nuclear genome, the marker DNA, preferably encoding a protein conferring a resistance to a herbicide on the plant; and 2) a female-fertile plant without the marker DNA in its genome.
U.S. Patent No.5,652,354 relates to promoters from endogenous genes of plants, wherein said promoters direct gene expression selectively in stamen cells of said plant, particularly in tapetum cells of said plant. The promoters may be used to transform a plant with a foreign DNA sequence encoding a product which selectively disrupts the metabolism, functioning, and/or development of stamen cells of the plant. The male-sterility DNA and its associated promoter are exemplified as being foreign DNA sequences. Preferred marker DNAs are those which inhibit or neutralize the action of herbicides.
None of these patents discloses a method of producing an open-pollinated population or synthetic variety whereby a transgene is maintained at sufficiently useful levels during subsequent generations of inter- and intra-crossing of the parental lines which made up the original population or variety.
It would be highly desirable to have a method to prevent the formation of, or eliminate, the individual seeds that do not carry a transgene. If this could be achieved, all the seeds in subsequent generations would carry a transgene, without interfering with the highly heterozygous genetic background of the cultivar. It would also offer a more convenient strategy for introgression of transgenes into open pollinated crop cultivars. A single transgenic individual could be intermated to many other individuals, with the high proportion of non-transgenic progeny being prevented from developing in seed production blocks prior to or during flowering and seed development.
Thus, the object of this invention to provide methods for producing segregating populations in which one or more transgenes are maintained in a large enough percentage of the plants so that the beneficial effect of the transgenes are realized.